I. Camptothecin (CPT) and Initial Clinical Trials
Camptothecin (CPT; IUPAC Nomenclature: (S)-4-Ethyl-4-hydroxy-1 H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)-dione) and certain of its analogs have been shown to possess varying degrees of anti-neoplastic activity. Presently, two CPT analogs (Irinotecan™ and Topotecan™, as discussed below) have been approved for therapeutic use in the United States by the Food and Drug Administration (FDA) for various forms of solid neoplasms.
CPT was initially isolated in 1966 by Wall, et al., from Camptotheca accuminata, (Nyssaceae family) a Chinese yew. See, Wall, M. E., et al., Plant chemotherapeutic agents. I. The Isolation and Structure of Camptothecin, a Novel Alkaloidal Leukemia and Tumor Inhibitor from Camptotheca Acuminata. J. Am. Chem. Soc. 88:3888-3890 (1966)).
The structure of this originally isolated camptothecin (CPT) is shown below:

By the early 1970's, CPT had reached Phase I and Phase II clinical trials and although it was found to possess anti-tumor activity, there were numerous deleterious physiological side-effects associated with its use. The side-effects included, but were not limited to, severe and unpredictable myelosuppression, gastrointestinal toxicity, hemorrhagic cystitis, alopecia, diarrhea, nausea, vomiting and the like. These toxicities, found during early clinical studies, rendered the drug “unmanageable” during this time period. See, Muggia, F. M.; et al., Phase I Clinical Trial of Weekly and Daily Treatment With Camptothecin (NSC-100880): Correlation With Preclinical Studies. Cancer Chemother. Rep. 56:515-521 (1972); Schaeppi, U., et al., Toxicity of Camptothecin (NSC-100880). Cancer Chemother. Rep. 5:25-36 (1974).
In order to demonstrate both the utility and novelty of the present invention, it will be instructive to engage in brief review of the published literature dealing with human clinical trials conducted with administered in a parenteral manner. Physicochemical studies of CPT found that the closed E-ring lactone form of CPT possessed extremely poor solubility in water (i.e., approximately 0.1 μg of drug dissolving in 1 mL of water). In addition, of the two CPT enantiomers, the naturally occurring (S)-isomer was found to be more potent than the (R)-isomer. See, e.g., Motwani, M. V., et al., Flavopiridol (Flavo) Potentiates the SN-38-Induced Apoptosis in Association with Downregulation of Cyclin Dependent Kinase Inhibitor p21waf1/cip1 in HCT116 Cells. Proc. Am. Assoc. Cancer Res. 41:32-43 (2000). These different properties of the various analogs are caused by the different chemical substituents on the core structure of CPT.
Thus, because of its extremely poor water solubility, in order for CPT to be administered in human clinical trials, it was initially formulated using sodium hydroxide. It is important to note, that all of these early clinical studies used sodium hydroxide formulations of CPT in order to markedly increase the water solubility (i.e., hydrophilicity) of the molecule to allow sufficient quantities of the agent to be administered parenterally to patients. The sodium hydroxide formulation of CPT created more water soluble CPT species that permitted clinicians to administer larger concentrations of CPT with smaller medication volumes of administration, thereby allowing sufficiently higher doses of the drug to be administered to cancer subjects undergoing Phase I and Phase II clinical trials. However, it was subsequently established that this formulation resulted in hydrolysis of the lactone E-ring of the camptothecin molecule, thus forming the water soluble carboxylate form of CPT which only possessed approximately one-tenth or less of the anti-tumor potency of the original, non-hydrolyzed lactone form of CPT. The clinical trials performed using the sodium hydroxide-formulated CPT provide to be highly disappointing, due to both the frequently-observed significant systemic toxicities and the lack of anti-neoplastic activity. It was subsequently ascertained that the drug's relative low hydrophilicity, was the most important reason for these side-effects. This low aqueous solubility of CPT in the lactone form greatly limited the practical clinical utility of the drug because prohibitively large volumes of fluid had to be administered to the subject in order to provide an effective dose of the drug. Because of the potent anti-neoplastic activity and poor water solubility of CPT lactone forms and many of its analogs in water, a great deal of effort was directed at generating new CPT lactone analogs that possessed greater aqueous solubility. Water soluble CPT analogs should not exist in large amounts in the open E-ring form but, alternately, should predominantly remain in the closed-ring lactone form, in order to be active. Thus, CPT analogs where equilibrium favors the closed-ring lactone form are desirable for administration.
II. Pharmacological Activity of CPT
Despite these earlier disappointing side-effects, increasing clinical interest in CPT was evoked during the 1980s, as a result of the revelation of its mechanism of action (i.e., Topoisomerase I inhibition). This new information regarding the mechanism of action of CPT analogs served to rekindle the interest in developing new Topo I inhibitors for use as anti-neoplastic drugs and subsequently several research groups began attempting to develop new CPT analogs for cancer therapy. See, Hsiang, Y. H., et al., Camptothecin Induces Protein-Linked DNA Breaks Via Mammalian DNA Topoisomerase I. J. Biol. Chem. 260:14873-14878 (1985); Hsiang, Y. H.; Liu, L. F., Identification of Mammalian DNA Topoisomerase I as an Intracellular Target of the Anticancer Drug Camptothecin. Cancer Res. 48:1722-1726 (1988); Hsiang, Y. H., et al., Arrest of Replication Forks by Drug-Stabilized Topoisomerase I DNA Cleavable Complexes as a Mechanism of Cell Killing by Camptothecin. Cancer Res. 49:5077-5082 (1989).
Several clinically important anticancer drugs kill tumor cells by affecting DNA Topoisomerases. Topoisomerases are essential nuclear enzymes that function in DNA replication and tertiary structural modifications (e.g., overwinding, underwinding, and catenation) which normally arise during replication, transcription, and perhaps other DNA processes. Two major Topoisomerases that are ubiquitous to all eukaryotic cells: (i) Topoisomerase I (Topo I) which cleaves single stranded DNA and (ii) Topoisomerase II (Topo II) which cleaves double stranded DNA. Topoisomerase I is involved in DNA replication; it relieves the torsional strain introduced ahead of the moving replication fork.
Topoisomerase I (Topo I) is a monomeric 100 kDal polypeptide containing 765 amino acids, and is encoded by a gene located on chromosome 20q12-13.2. See, e.g., Creemers, G. J., et al., Topoisomerase I Inhibitors: Topotecan and Irinotecan. Cancer Treat. Rev. 20:73-96 (1994); Takimoto, C. H.; Arbuck, S. G. The Camptothecins. Cancer Chemother and Biother. 2nd edition (B. L. Chabner, D. L. Longo (eds)), 463-384 (1996). It is an essential enzyme in DNA replication and RNA transcription, and is present in all eukaryotic (including tumor) cells. Since normal DNA is super-coiled, and tightly fitted in the chromosomes, the DNA-replication fork is unable to synthesize new DNA out of this topological constrained DNA. Topo I acts in an ATP-independent fashion, by binding to super-coiled DNA and cleaving a phosphodiester bond, resulting in a single-strand break. At the same time, Topo I forms a covalent reversible adduct between a tyrosine residue at position 723 of Topo I and the 3′ end of the single-strand DNA molecule, called the cleavable complex. The DNA molecule is able to rotate freely around the intact single DNA strand, and relaxation of the DNA occurs. After the religation of the cleavage, Topo I dissociates from the DNA. The cleavable complex usually is present for only a short time, just to allow the single uncleaved DNA strand to unwind.
Specifically, it was found that CPT forms a reversible covalent complex comprising: Topo I-CPT-DNA. In brief, the primary mechanism of action of CPT is the inhibition of Topo I by blocking the rejoining step of the cleavage/relegation reaction of Topo I, thus resulting in the accumulation of covalent reaction intermediates (i.e., the cleavable complex). CPT-based cellular apoptosis is S-phase-specific killing through potentially lethal collisions between advancing replication forks and Topo I DNA complexes. Two repair responses to Topo I-mediated DNA damage involving covalent modification of Topo I have been identified. The first involves activation of the Ubiquitin/26S proteasome pathway, leading to degradation of Topo I (CPT-induced Topo I down-regulation). The second involves the Small Ubiquitin-like Modifier (SUMO) conjugation to Topo I. These repair mechanisms for Topo I-mediated DNA damage play an important role in determining CPT sensitivity/resistance in tumor cells.
Topo I purified from human colon carcinoma cells or calf thymus has been shown to be inhibited by CPT. CPT, Irinotecan™ (CPT-11) and an additional Topo I inhibitor, Topotecan, has been in used in clinical trials to treat certain types of human cancer. For the purpose of this invention, CPT analogs include: 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxy camptothecin (Irinotecan™ or CPT-11), 10-hydroxy-7-ethyl camptothecin (HECPT), 9-aminocamptothecin, 10,11 methylenedioxy camptothecin and 9-dimethylaminomethyl-10-hydroxy camptothecin (Topotecan). These CPT analogs use the same mechanism to inhibit Topo I; they stabilize the covalent complex of enzyme and strand-cleaved DNA, which is an intermediate in the catalytic mechanism. These analogs have no binding affinity for either isolated DNA or Topo I but do bind with measurable affinity to the enzyme-DNA complex. The stabilization of the Topo I “cleavable complex” by CPT and analogs is readily reversible.
Topoisomerase II (Topo II) works in a similar way to Topo I, with the difference being that the former enzyme acts ATP-dependently, to cause reversible doublestrand DNA cleavage, in the relaxation of DNA. Direct interference of CPTs with Topo II has not been described. However, it has been reported that Irinotecan™ (CPT-11) treatment sensitizes some tumor-xenografts in mice to Topo II inhibitors, by increasing the Topo II mRNA expression after 24 and 48 hours. This suggests that combination therapies with Topo I and Topo II targeting chemotherapy for human solid tumors might be valuable. The CPT analogs inhibit the religation reaction of Topo I by selectively inducing a stabilization of the cleavable complexes at Topo I sites bearing a guanine residue at the 5′-terminus of the enzyme mediated breaks. See, e.g., Svejstrup, J. Q., et al., Technique for Uncoupling the Cleavage and Religation Reactions of Eukaryotic Topoisomerase I. The Mode of Action of Camptothecin at a Specific Recognition Site. J. Mol. Biol. 222:669-678 (1991); Jaxel, C., et al., Effect of Local DNA Sequence on Topoisomerase I Cleavage in the Presence or Absence of Camptothecin. J. Biol. Chem. 266:20418-20423 (1991); Tanizawa, A., et al., Induction of Cleavage in Topoisomerase I c-DNA by Topoisomerase I Enzymes From Calf Thymus and Wheat Germ in the Presence and Absence of Camptothecin. Nucl. Acids Res. 21:5157-5166 (1994). Although this stabilization in itself is reversible, an irreversible doublestrand break occurs when a replication fork meets a cleavable complex. The higher the levels of Topo I, the higher the frequency of cleavable complexes, and the higher the number of DNA breaks. These breaks may lead to cell cycle arrest in the S/G2-phase, activation of apoptosis pathways, and finally to cell death. See, e.g., Hsiang, Y. H., et al., Arrest of Replication Forks by Drug-Stabilized Topoisomerase I DNA Cleavable Complexes as a Mechanism of Cell Killing by Camptothecin. Cancer Res. 49:5077-5082 (1989). As a result of this, Topo I inhibitors are only lethal in the presence of ongoing DNA replication or RNA transcription. See, e.g., D'Arpa, P., et al., Involvement of Nucleic Acid Synthesis in Cell Killing Mechanisms of Topoisomerase I Poisons. Cancer Res. 50:6919-6924 (1990). S-phase synchronized cells appeared to be much more sensitive to Topo I inhibitors, compared to G1- or G2/M-cells, suggesting an S-phase specific cytotoxicity for this type of drugs. See, e.g., Takimoto, C. H., et al., Phase I and Pharmacologic Study of Irinotecan Administered as a 96-Hour Infusion Weekly to Adult Cancer Patients. J. Clin. Oncol. 18:659-667 (2000). In colon, prostate, ovary and esophagus tumors, elevated Topo I levels have been found, whereas in kidney tumors and non-Hodgkin lymphomas this was not the case See, e.g., Van der Zee, A., et al., P-glycoprotein Expression and DNA Topoisomerase I and II Activity in Benign Tumors of the Ovary and in Malignant Tumors of the Ovary, Before and After Platinum/Cyclophosphamide Chemotherapy. Cancer Res. 51: 5915-5920 (1991). Recent investigations have indicated that Irinotecan™ and Topotecan are also inhibitors of angiogenesis, a property that might contribute to their chemotherapeutic activity. Neovascularization has been positively correlated with increasing invasion and metastases of various human tumors. In mice cornea models, anti-angiogenic effects of some CPTs, including Irinotecan™ (CPT-11), were studied. Angiogenesis was induced by fibroblast growth factor, but by increasing the dose of Irinotecan™ CPT-11, the area of angiogenesis in the tumor decreased, following a negative, almost exponential, curve. At dose levels of 210 mg/kg a significant reduction of neovascularization was observed.
Although CPT and the aforementioned CPT analogs have no discernable direct effects on Topo II, these CPT analogs are believed to stabilize the “cleavable complex” in a manner analogous to the way in which epipodophyllotoxin glycosides and various anthracyclines inhibit Topo II.
Inhibition of Topo I by CPT and analogs induces protein-associated-DNA single-strand breaks. Virtually all of the DNA strand breaks observed in vitro cells treated with CPT are protein linked. However, an increase in unexplained protein-free breaks can be detected in L1210 cells treated with CPT. The analogs appear to produce identical DNA cleavage patterns in end-labeled linear DNA. It has not been demonstrated that CPT or CPT analogs cleaves DNA in the absence of the Topo I enzyme.
III. Cell Cycle-Specific Activity of Camptothecin
The activity of CPT is cell cycle-specific. The greatest quantitative biochemical effect observed in cells exposed to CPT is DNA single-strand breaks that occur during the S-phase. Because the S-phase is a relatively short phase of the cell cycle, longer exposure to the drugs results in increased cell killing. Brief exposure of tumor cells to the drugs produces little or no cell killing, and quiescent cells are refractory. These aforementioned results are likely due to two factors:                (i) This class of drugs inhibit the normal activity of Topo I, reversibly. Although they may produce potentially lethal modifications of the DNA structure during DNA replication, the DNA strand breaks may be repaired after washout of the drug; and        (ii) Cells treated with Topo I inhibitors, such as CPT tend to stay in G0 of the cell cycle until the drug is removed and the cleaved DNA is repaired. Inhibitors of these enzymes can affect many aspects of cell metabolism including replication, transcription, recombination, and chromosomal segregation.IV. Previously-Tested Camptothecin Analogs        
As discussed above, CPT and many of its analogs (see e.g., Wall and Wani, Camptothecin and Taxol: Discovery to Clinic-Thirteenth Bruce F. Cain Memorial Award Lecture Cancer Research 55:753-760 (1995)) are poorly water soluble and are reportedly also poorly soluble in a number of pharmaceutically-acceptable organic solvents as well. However, there are numerous reports of newly created water soluble analogs of CPT (Sawada, S., et al., Synthesis and Antitumor Activity of Novel Water Soluble Analogs of Camptothecin as Specific Inhibitors of Topoisomerase I. Jour. Med. Chem. 38:395-401 (1995)) which have been synthesized in an attempt to overcome some of the significant technical problems in drug administration of poorly water soluble camptothecins to subjects with cancer. Several water soluble CPT analogs have been synthesized in an attempt to address the poor water solubility and difficulties in administration to subjects. Several examples of these water soluble CPT analogs are set forth below in Table I:
TABLE I9-dimethylaminomethyl-10-hydroxycamptothecin (Topotecan)7-[(4-methylpiperazino)methyl]-10,11-ethylenedioxycamptothecin7-[(4-methylpiperazino)methyl]-10,11-methylenedioxycamptothecin7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin(Irinotecan or CPT-11)9-nitrocamptothecin (Rubitecan)
Other substituted CPT analogs with different solubility and pharmacologic properties have been synthesized as well; examples of these camptothecin analogs include 9-aminocamptothecin and 9-nitrocamptothecin (Rubitecan) that are poorly soluble in both aqueous and non-aqueous media and have been tested in humans. Rubitecan (9-nitrocamptothecin) is a prodrug of 9-aminocamptothecin, and has been shown to spontaneously convert to 9-aminocamptothecin in aqueous media and in vivo in mice, dogs and humans (see, Hinz, et al., Pharmacokinetics of the in vivo and in vitro Conversion of 9-Nitro-20(S)-camptothecin to 9-Amino-20(S)-camptothecin in Humans, Dogs and Mice, Cancer Res. 54:3096-3100 (1994)).
The pharmacokinetic behavior of 9-nitrocamptothecin and 9-aminocamptothecin is similar to the water-soluble camptothecin analogs (i.e., Topotecan and Irinotecan™) in that the plasma half lives are markedly shorter than the more lipid soluble CPT analogs. An additional major problem with 9-aminocamptothecin is that its chemical synthesis using the semi-synthetic method is performed by nitration of CPT, followed by reduction to the amino group, which is a very low yield type of synthesis. 9-aminocamptothecin is also light sensitive, heat sensitive and oxygen sensitive which render both the initial synthesis and subsequent stability (i.e., shelf-life) of 9-aminocamptothecin problematic, at best. Moreover, the chemical decomposition reactions of 9-aminocamptothecin frequently result in the formation of analogs that exhibit a large degree of toxicity in nude mice, whereas pure 9-aminocamptothecin is significantly less toxic.
As previously discussed, 9-aminocamptothecin is also difficult to administer to subjects because it is poorly soluble in both aqueous and organic solvents. Alternately, while 9-nitrocamptothecin is easier to produce and is more chemically stable, the chemical conversion to 9-aminocamptothecin causes the drug is reportedly susceptible to MDR/MRP tumor-mediated drug resistance, which further limits its utility in the unfortunately common setting of drug resistant neoplasms. Based on pharmacokinetic behavior and chemical properties, 9-aminocamptothecin is predicted to have reduced tissue penetration and retention relative to more lipid soluble camptothecin analogs. Further, its poor solubility diminishes the amount of the drug that can cross the blood/brain barrier.
Of this diverse group of substituted CPT analogs undergoing human clinical development, Irinotecan™ (CPT-11) has been one of the most extensively studied in both Phase I and Phase II clinical trials in human patients with cancer. It is noteworthy that 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxy camptothecin (Irinotecan™), which is a water soluble prodrug, is biologically inactive and requires activation by a putative carboxylesterase enzyme. The active species of Irinotecan™ is the depiperidenylated 10-hydroxy-7-ethyl camptothecin (as claimed in Miyasaka, et al., U.S. Pat. No. 4,473,692, (1984)), which is also known as SN38. SN38 is a toxic lipophilic metabolite, which is formed by an in vivo bioactivation of Irinotecan™ by a putative carboxylesterase enzyme.
SN38 is very poorly soluble in water and has not been directly administered to human patients with cancer. Recently, it has been reported in human patients that SN38 undergoes further metabolism to form a glucuronide species, which is an inactive form of the drug with respect to anti-tumor activity, and also appears to be involved in producing human toxicity (e.g., diarrhea, leukopenia) and substantial interpatient variability in drug levels of the free metabolite and its glucuronide conjugate.
Irinotecan™ has been tested in human clinical trials in the United States, Europe and Japan. Clinical studies in Japan alone, have reported approximately 100 patient deaths which have been directly attributable to Irinotecan™ drug toxicity. The Miyasaka, et al. patents (U.S. Pat. No. 4,473,692 and U.S. Pat. No. 4,604,463) state that the object of their invention is to “ . . . provide 10-substituted camptothecins which are strong in anti-tumor activity and possess good absorbability in living bodies with very low toxicity” and “ . . . to provide new camptothecin analogs which are strong in anti-tumor activity and possess good solubility in water and an extremely low toxicity”.
Having multiple drug-related human deaths and serious patient toxicity, is clearly a failure of the aforementioned 10-substituted camptothecins synthesized by Miyasaka, et al., to fulfill their stated objectives. It is notable that tremendous interpatient variability with regard to drug levels of various forms, drug metabolism, certain pharmacokinetic properties and toxicity has been reported with the use of Irinotecan™ in human subjects with cancer. Parenteral administration of Irinotecan™ can achieve micromolar plasma concentrations of Irinotecan™ that, through metabolism to form SN38, can yield nanomolar concentrations of the active metabolite SN38. It has recently been reported in human subjects that SN38 undergoes further metabolism to form the SN38 glucuronide (see, e.g., Gupta, et al., Metabolic Fate of Irinotecan in Humans: Correlation of Glucuronidation with Diarrhea. Cancer Res. 54:3723-3725 (1994)).
This further metabolic conversion of Irinotecan™ is important, since there is also reportedly large variability in the conversion of Irinotecan™ to SN38 and large interpatient variability in the metabolism of SN38 to form the inactive (and toxic) SN38 glucuronide conjugate in human subjects. (see, e.g., Gupta, et al., Metabolic Fate of Irinotecan in Humans: Correlation of Glucuronidation with Diarrhea. Cancer Res. 54:3723-3725 (1994) and Ohe, et al., Phase I Study and Pharmacokinetics of CPT-11 with 5-Day Continuous Infusion. JNCI 84(12):972-974 (1992)).
Since the amount of Irinotecan™ and SN38 metabolized is not predictable in individual patients, significant clinical limitations are posed and create the risk of life-threatening drug toxicity, and/or risk of drug inactivity due to five putative biological mechanisms: (i) conversion of greater amounts of Irinotecan™ to SN38; (ii) inactivation of SN38 by glucuronidation; (iii) conversion of SN38 glucuronide to free SN38; (iv) lack of anti-neoplastic activity due to the conversion of lesser amounts of Irinotecan™ to form SN38; and (v) lack of anti-neoplastic activity by more rapid and extensive conversion of SN38 to form the glucuronide species. It is important to note that even a doubling of the plasma concentration of the potent Irinotecan™ metabolite SN38 may result in significant toxicity, because free SN38 exhibits anti-neoplastic activity at nanomolar concentrations.
Another source of interpatient variability and toxicity is the in vivo de-glucuronidation of SN38 and similar CPT analogs to produce a free and active species of the drug. Deglucuronidation of a CPT analog that is susceptible to A-ring glucuronidation, such as SN38, results in an increase in the plasma or local tissue concentration of the free and active form of the drug, and if high enough levels were reached, patient toxicity, and even death may result.
In addition to the two aforementioned FDA-approved drugs, there are currently at least nine camptothecin analogs that have been evaluated in various stages of clinical testing. These camptothecin analogs include:
1. Karenitecin (BNP1350)
BNP1350 is a highly lipophilic camptothecin analog having a 7-trimethylsilylethyl moiety and is claimed in U.S. Pat. No. 5,910,491, along with formulations and uses thereof. Formulations of BNP1350 with the solvent N-methylpyrrolidinone (NMP) are claimed in, e.g., U.S. Pat. No. 5,726,181.
2. Lurtotecan (NX 211)
NX211 is a water-soluble camptothecin having a 10,11-ethylenedioxy moiety and a cleavable 4-methylpiperazino methyl moiety at C7. By way of example, U.S. Pat. No. 5,559,235 discloses and claims the analogs and formulations, and uses thereof.
3. Exatecan (DX-8951f)
DX-8951f is a hexacyclic camptothecin analog, having 10-methyl and 11-fluoro substitutions, and with its sixth ring fused between C7 and C9. By way of example, and not of limitation, U.S. Pat. No. 5,637,770 describes and claims the analog, and formulations and uses thereof.
4. Diflomotecan (BN 80915)
BN 80915 is a 10,11-difluorocamptothecin, with a 7-member E-ring. By way of example, and not of limitation, U.S. Pat. No. 5,981,542 describes and claims the analog, and its uses and formulations.
5. Rubitecan (9-Nitro CPT)
9-Nitrocamptothecin, as mentioned above is poorly soluble in both aqueous and organic solvents and is described and is not claimed any United States Patents, with the first publication of the analog occurring in Japanese Patent Application No. 82-160944 in 1982. Several patents have issued since then, all regarding processes for preparing the analog as well as uses thereof.
6. Afeletecan (CPT Glycoconjugate)
Afeletecan is an C20 glycoconjugated, water-soluble analog of camptothecin and is described and claimed in U.S. Pat. No. 6,492,335.
7. Gimatecan (ST 1481)
ST1481 is a water-soluble prodrug having a C7 imino moiety, bonded to a terminal tert-butoxy group. The analog is described and claimed in U.S. Pat. No. 6,242,257.
8. Mureletecan (PNU 166148)
Mureletecan is another water-soluble prodrug having a cleavable peptide moiety bonded to C20 to form an ester.
9. Pegbetotecan, Pegcamotecan, Peglinxotecan (PEG CPT; Prothecan®)
This prodrug includes a cleavable water-soluble polyethylene glycol moiety that forms an ester at C20. By way of example, the analog is described and claimed in U.S. Pat. No. 5,840,900.
The various chemical structures of the nine aforementioned camptothecin analogs are set forth in Table II, below:
TABLE II       
Poorly water-soluble (i.e., hydrophobic) camptothecins are necessarily formulated for administration by dissolution or suspension in organic solvents. U.S. Pat. Nos. 5,447,936; 5,726,181; 5,859,022; 5,859,023; 5,880,133; 5,900,419; 5,935,967; 5,955,467; and other describe pharmaceutical formulations of highly lipophilic, poorly water-soluble camptothecin analogs in various organic solvents, namely N,N-dimethylacetamide (DMA); N,N-dimethylisosorbide (DMI); and N-methylpyrrolidinone (NMP).
V. Formulation and Administration of CPT and Analogs
In the early-1970's, clinical studies utilizing the sodium salt of camptothecin were begun at the Baltimore Cancer Research Center. In this clinical trial, CPT was administered as a rapidly running IV solution over a 5-10 minute period at a concentration of 2 mg of camptothecin sodium per milliliter of saline. Doses of CPT sodium from 0.5 to 10.0 mg/kg of actual or ideal body weight (whichever was less) were used. These investigators reported that because hemorrhagic sterile cystitis was noted in several of the early trials, patients receiving camptothecin sodium were well-hydrated either intravenously (i.v.) or orally for 72 hours after drug administration. It is noteworthy that the mean urine recovery of CPT was 17.4% over the first 48 hours (with the range from: 3.6% to 38.9%) with most of the excretion occurring in the initial 12 hours. When these investigators excluded the five patients with impaired excretion, the mean urine recovery of CPT was 22.8%. These investigators noted that non-metabolized camptothecin in high concentrations rapidly appeared in the urine after iv drug administration and went further to state that this finding probably accounted for the sterile hemorrhagic cystitis noted in three moderately dehydrated patients. Although maintaining a copious urine outflow seems able to prevent this complication, the investigators reported that they were exploring various alterations in urine pH as another possible way of decreasing the risk of this debilitating type of toxicity.
Muggia, et. al. (Phase I Clinical Trial of Weekly and Daily Treatment with Camptothecin (NSC-100880): Correlation with Preclinical Studies. Cancer Chemotherapy Reports, Part 1. 56(4):515-521 (1972)) reported results of a Phase I clinical trial in fifteen patients treated with CPT sodium at four weekly dose levels ranging from 20-67 mg/m2. No clinical benefit was observed in eight patients with measurable disease who were treated with the 5-day courses at dose levels associated with toxicity. The CPT was administered in concentrations of 1 or 10 mg/mL and it was always administered by intravenous push. Cystitis was the most prominent non-hematologic toxic effect observed in this study. Bladder toxicity was dose limiting in three patients receiving doses of 20 to 30 mg/m2, and occurred in two additional patients at doses of 30 and 44 mg/m2. Cystitis, another toxic effect occurring frequently after treatment with camptothecin, was not predicted by preclinical toxicological studies. Clinical experience present inventors would suggest that the occurrence of cystitis may be related to the duration of the patient's exposure to the drug. It is their experience that CPT is excreted unchanged by the kidneys, although a large percentage of the drug administered cannot be accounted for in the urine. It is possible that relatively less drug is excreted in the urine of animals since an extremely active transport of CPT into bile has been demonstrated. Alternatively, one needs to postulate that the mucosa of the human bladder is more susceptible to the toxic action of CPT or that the effect on the human bladder is due to some unrecognized CPT metabolite.
In 1972, Moertel and coworkers administered CPT sodium dissolved in physiologic saline at a concentration of 2 mg/mL and administered by rapid intravenous infusion over 5-10 minutes. Two schedules of administration were used in this study: (i) a single injection repeated at 3-week intervals; and (ii) a 5-day course repeated every 4 weeks. The initial dose for the single-dose method was 180 mg/m2. Because of toxic effects, which were considered excessive by the investigators, later patients were treated at doses ranging between 90 and 120 mg/m2. Dosages for the 5-day course ranged between 11 and 22 mg/m2/day (total course: 55-110 mg/m2). The toxicity and response data from this aforementioned study is summarized, below, in Table III-Table VI. Diarrhea was only a problem at higher doses, although it could be quite severe to the point of fecal incontinence and could persist for as long as 4 weeks. Cystitis usually began about 7-10 days after treatment and was characterized clinically by dysuria and frequency. With more severe toxicity, gross hematuria developed. Pathologically, this was characterized by multiple necrotic ulcerations which could involve the entire urinary tract from kidney pelvis to bladder. According to these investigators, the occurrence of hemorrhagic cystitis did not preclude further treatment with CPT, and its severity could be titrated down by lowering the dose in subsequent courses. These investigators also reported that the more prolonged schedule produced more severe toxicity at a given total dose level, but the difference was not as great as might have been predicted by preclinical animal studies.
These investigators proposed that a reasonable initial dose of CPT sodium is 110-120 mg/m2 for the single-injection method or 17 mg/m2/day (total dose: 85 mg/m2) for the 5-day course. They noted that after 2 months (8 or 9 weeks) only two of their 61 patients showed evidence of partial objective improvement and none showed improvement at 3 months. Both patients who demonstrated an objective response at 2 months had large bowel cancer. These investigators concluded that CPT “ . . . is a drug of protean and unpredictable toxicity that has no clinical value in the management of gastrointestinal cancer.”
TABLE IIIToxic Reactions: Single-Dose MethodNumber of Patients with Non-Hematologic Toxicity:No. ofDosePatients(mg/m2)TreatedDiarrheaCystitis901010062110211120742180923
TABLE IVToxic Reactions: 5-day CourseNon-Hematologic Toxicity No. of Patients With:No. of PatientsDose (mg/m2 × 5)TreatedDiarrheaCystitis112115914175422010462211
TABLE VRelationship of Method of Administration to CystitisMethod of AdministrationSingle Dose5-Day CourseCystitis(% of 34 Patients)(% of 27 Patients)2448 (P < 0.05)
TABLE VIObjective ResultsSingle-Dose Method (34 Patients Total)Time afterstart of therapyObjective Results*3 wks6 wks9 wks12 wksImproved422—Stable17118 6Worse132124285-Day Course (27 Patients Total)Time afterstart of therapyObjective results*4 wks8 wks12 wksImproved1——Stable12 7 6Worse142021*A total of 3 patients showed a 25%-50% response at 3 wks, only.
In another study, Gottlieb and Luce (Treatment of Malignant Melanoma with Camptothecin (NSC-100880) Cancer Chemotherapy Reports, Part 1 56(1):103-105 (1972)) reported the results of treatment of patients with malignant melanoma with CPT sodium (1972). Fifteen patients with advanced malignant melanoma were treated with CPT at doses of 90-360 mg/m2 repeated every 2 weeks. CPT-sodium was administered as a single rapid intravenous (IV) injection starting at a dose of 120 mg/m2 repeated at 2-week intervals. The dose in subsequent courses was increased by increments of 60 mg/m2 per dose (to a maximum of 360 mg/m2) in eight patients who tolerated their initial doses with minimal toxicity. To prevent the known bladder toxicity of this drug, patients were well hydrated for 3 days after therapy. None of the patients had a 50% or greater decrease in tumor diameter. Less pronounced transient tumor regression was noted in three patients, but no clinical benefit was associated with these responses. The remaining patients had no change or progression in their disease. Toxic effects included myelosuppression (11 patients), nausea and vomiting, alopecia, diarrhea, and hemorrhagic cystitis. These investigators concluded that CPT, at least as administered in this study, had little to offer the patient with advanced disseminated melanoma.
Creaven, et al., (Plasma Camptothecin (NSC-100880) Levels During a 5-Day Course of Treatment: Relation to Dose and Toxicity. Cancer Chemotherapy Reports Part 1 56(5):573-578 (1979)) reported studies of plasma CPT levels during a 5-day course of treatment. These investigators state that the toxicity of CPT has been widely and unpredictably variable in the course of initial clinical evaluation. Severe toxic effects occurred even though patients with obvious renal disease were excluded. In this study they investigated plasma CPT levels 24 hours after the administration of sodium CPT administered on a once daily over a 5 day total schedule to determine whether such measurements would be of value in predicting toxicity, and observed that plasma CPT levels have little relation to the dose given when the dose is in the range of 6.5-20 mg/m2/day.
There are several features which establish a commonality with these aforementioned studies with those utilizing sodium CPT. First, is the use of sodium-CPT which made the CPT more water soluble by hydrolysis of lactone E ring to form the carboxylate species (i.e., by formulating CPT in sodium hydroxide). The anti-tumor activity of the carboxylate form of CPT is reduced by at least 10-fold, which partially accounts for the lack of clinical response in these studies. Second, is the rapid intravenous administration of the drug. CPT is an S-phase specific drug and therefore will exert a greater chemotherapeutic effect under conditions of prolonged exposure, as in a continuous intravenous infusion. The short infusion (i.v. “push” or rapid i.v. infusion) times in all of these studies do not allow a long enough exposure time to the drug at suitable levels, and is further compounded by the administration of the water soluble carboxylate form of CPT. A third common feature is the notable frequency of cystitis in these studies using sodium CPT.
VI. Lactone Form Stabilizes CPT Anti-Tumor Activity and Reduces Water Solubility
Utilizing HPLC and NMR techniques, researchers have demonstrated that CPT analogs undergo an alkaline, pH-dependent hydrolysis of the E-ring lactone. The slow reaction kinetics allow one to assess whether both the lactone and non-lactone forms of the drug stabilizes the Topo I-cleaved DNA complex. Studies indicate that only the closed lactone form of the drug helps stabilize the cleavable complex. This observation provides reasoning for the high degree of CPT activity observed in solid tumor models. Tumor cells, particularly hypoxic cells prevalent in solid neoplasms, have lower intracellular pH levels than normal cells. At pH levels below 7.0, the closed form of CPT predominates. Thus, the present inventors maintain that CPT will be more effective at inhibiting Topo I in an acidic environment than in cells having higher intracellular pH levels. It is the object of the instant invention to provide lactone stable CPT as the basis of the claimed subject matter. Lactone-stable CPT is defined as CPT which is dissolved in DMI or DMA in the presence of a pharmaceutically-acceptable acid. The presence of the acid stabilizes the lactone form of CPT.
While some authors have stated that the anti-neoplastic effects of CPT is inversely proportional to the molecules hydrophilicity, this cannot state this with certainty because 9-nitrocamptothecin (9-NC) and 9-aminocamptothecin (9-AC) are neither hydrophilic or lipophilic, and no significant antitumor activity has been demonstrated with either one of these compounds. Thus, while water solubility is not required for antitumor activity, a lactone configuration or the equivalent appears to be required for improved antitumor activity of all camptothecins. For example, the lactone form of CPT is poorly soluble in water and yet has significant anti-tumor activity. Thus, providing evidence that the hydrolysis of E-ring lactone to the carboxylate form of CPT greatly increases the water solubility of molecule at the expense of significantly reducing its anti-tumor activity. As previously mentioned, the novel, “flipped” E-ring camptothecin analogs of the present invention belong to a class of drugs with a pH- and protein-dependent interconversion between the pharmacologically-active α-hydroxy-δ-lactone form (i.e., lactone form) and the inactive α-hydroxy-δ-carboxylate form (i.e., carboxyl form), which necessitates analysis of blood samples (i.e., rapid centrifugation to collect the plasma). For example, when total concentrations (i.e., α-hydroxy-δ-lactone+α-hydroxy-δ-carboxylate) are to be measured, this rapid blood processing is crucial, since only the lactone form is able to diffuse across the cell membranes of erythrocytes, and thus a change in the lactone form (i.e., α-hydroxy-δ-lactone) to carboxylate form (i.e., α-hydroxy-δ-carboxylate) ratio has an effect on the total drug concentrations in the plasma compartment. Stabilization of the lactone to carboxylate ratio by direct freezing of the plasma sample is the most convenient approach for the determination of the α-hydroxy-δ-lactone only concentrations. At the time of analysis, all samples can be handled at once by solid-phase or liquid-liquid extraction techniques, in which only the lipophilic lactone form is extracted, while the inactive α-hydroxy-δ-lactone form is discarded. The total drug concentrations are measured in a second analysis after acidification of the samples. A second simple way of stabilizing the lactone to carboxylate ratio is by cold methanolic deproteinization of plasma samples, immediately after collection of the plasma. An advantage of this procedure is the possibility of measuring the α-hydroxy-δ-lactone and α-hydroxy-δ-carboxylate forms simultaneously in one analytical run. Quantification of the CPTs is usually carried out by reversed phase high-performance liquid chromatography (HPLC), and since most CPT analogs have strong fluorescence characteristics, fluorescence detection is the most frequently applied detection technique. See, e.g., Loos, W. J., et al., Determination of Camptothecin Analogs in Biological Matrices by High-Performance Liquid Chromatography. Anticancer Drugs 11:315-324 (2000).
A non-enzymatic, pH-dependent, reversible interconversion exists between two forms of the six-membered E-ring camptothecin analogs. The first form, is the pharmacologically-active, novel, “flipped” α-hydroxy-δ-lactone, E-ring form of the present invention (which predominates at acidic pH), wherein the E-ring is in the closed orientation. The second form, is the pharmacologically-inactive α-hydroxy-δ-carboxylate, E-ring form of the present invention (which predominates at basic pH), wherein the E-ring is in the open orientation. The inactive, “open” α-hydroxy-δ-carboxylate form not only lacks the ability to stabilize the cleavable complex, but it is also not able to passively diffuse across cell membranes and is thus pharmacologically-inactive. The equilibrium between the “closed” α-hydroxy-δ-lactone form and the “open” α-hydroxy-δ-carboxylate form is not only dependent upon the pH of the solution, but also upon different affinities of the two different species for proteins. For example, CPT has shown to be inactivated by human serum albumin, by a preferential binding of the carboxylate form to human serum albumin, resulting in a shift of the equilibrium towards the carboxylate form. See, Loos, W. J., et al., A. Role of Erythrocytes and Serum Proteins in the Kinetic Profile of Total 9-Amino-20(S)-Camptothecin in Humans. Anticancer Drugs 10:705-7109 (1999).
Various investigators have claimed that reason previous use of sodium hydroxide-treated CPT (sodium-CPT) caused hemorrhagic cystitis relates to the enhanced renal excretion of the carboxylate form of CPT which when exposed to the lower pH (˜pH 5) of the distal convoluted tubule in the kidney, the carboxylate form of CPT is converted to the lactone form of CPT. The formation of the lactone form in high concentration at the distal convoluted tubule resulted in a high concentration of the lactone form of CPT being excreted into the collecting duct and into the ureters and bladder which resulted in hemorrhagic cystitis. Elimination of CPT by the renal route is enhanced by administration of the carboxylate form and such elimination may be reduced by administration of the CPT lactone form.
Two CPT analogs (i.e., CPT-11 and Topotecan), have less sporadic toxicities but retain significant activity of the parent analog. CPT-11 and Topotecan are currently approved for human use by the U.S. Food and Drug Administration in the United States. In addition, 10,11-methylenedioxycamptothecin is reportedly very active in preclinical studies, but it is also reported to be relatively insoluble in water which limits its use in the clinic.
Table VII, below, presents data summarizing Phase I clinical trials of CPT-11. Neutropenia and diarrhea were the major reported, dose-limiting toxicities of CPT-11.
TABLE VIIPHASE I STUDIES CPT-11InvestigatorSchedule# Pts.DoseToxicityTumor TypeClavel, et al90 min.37115 mg/m2/dneutropenia*Breast(1 PR)QDx 3 Q21 (33-115)diarrhea,MesotheliomaDaysNausea and(1 PR)vomiting,Culine, et al90 min.59150 mg/m2/wkneutropenia*EsophagusQ21 days (50-150)diarrhea*(1 PR) cervixvomiting,(1 PR) renalAlopecia(1 PR)OvarianFatigue(1 PR)Stomatitisneutropenia*Negoro, et al30 min17100 mg/m2diarrhea*, N/V,NS CLC(2 PRs)Infusion (50-150)alopecia,WeeklyLiverDysfunctionOhe, et al120 hr CI36 40 mg/m2/ddiarrhea*NoneQ3 wks (5-40)Nausea andvomiting,thrombocytopeniaAnemia, liverDysfunctionRothenberg, et al90 mg QWx 432180 mg/m2/wkneutropenia,Colon CaQ42 days (50-180)Nausea,(2 PRs)vomiting,AlopeciaRowinsky, et al90 min32240 mg/m2neutropenia*ColonCancer(1 PR)Infusion(100-345)vomiting,Cervix CaQ21 daydiarrhea abd.(1 PR)pain, flushing*Dose Limiting Toxicity
It should be noted that the analogs disclosed in the present invention, which can be administered either orally or parenterally to patients substantially in the lactone form, which greatly ameliorate renal elimination of CPT and the concomitant incidence of hemorrhagic cystitis. In addition to the aforementioned toxicities and limited clinical responses to previously-described CPTs, CPT has also been considered unsuitable for direct clinical use because it is poorly soluble in water. One useful purpose of the present invention is to formulate CPT in a pharmaceutically-acceptable manner to stabilize CPT in the lactone ring form. It is this lactone stable CPT formulation which permits direct administration of CPT to cancer patients. An additional purpose of this invention to provide certain indications, schedules, dosages and routes of administration of a lactone-stable camptothecin for the purpose of treating cancer in humans. Another advantage of present invention provides clinicians with the ability to directly adjust the plasma levels of CPT to the point of therapeutic tolerance by controlling the dose and the schedule of administration. The present inventors contend that this should lead to a superior ability to achieve better chemotherapeutic activity and reduce interpatient variability of the plasma levels of CPT.
As previously discussed, a lactone-stable form of CPT has not been administered by parenteral or oral routes in human subjects for the purpose of inhibiting the growth of cancer cells. The present invention overcomes these limitations and discloses and claims: (i) a novel, lactone-stable, “flipped” E-ring camptothecin, pharmaceutically-acceptable salts, and/or analogs thereof; (ii) methods of synthesis of said novel, lactone-stable, “flipped” E-ring camptothecin, pharmaceutically-acceptable salts, and/or analogs thereof; (iii) pharmaceutically-acceptable formulations comprising said novel, lactone-stable, “flipped” E-ring camptothecin, pharmaceutically-acceptable salts, and/or analogs thereof, and, optionally, one or more additional chemotherapeutic agents; (iv) methods of administration of said novel, lactone-stable, “flipped” E-ring camptothecin, pharmaceutically-acceptable salts, and/or analogs thereof, and, optionally, one or more additional chemotherapeutic agents, to subjects in need thereof; and (v) devices for the administration of said novel, lactone-stable, “flipped” E-ring camptothecin, pharmaceutically-acceptable salts, and/or analogs thereof, and, optionally, one or more chemotherapeutic agents, to subjects in need thereof.